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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
• • C—CHEMISTRY; METALLURGY
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus
  • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

• This invention relates to a new amylase-negative mutant of Bacillus subtilis useful as a host into which vectors containing amylase-coding genes can be introduced using recombinant DNA methodology.
• Most genetic material in a bacterium exists as giant DNA molecules which are present as the chromosome of the cell. A certain amount of the genetic material may also be present in the form of smaller, closed circular DNA molecules known as plasmids. The portion of the DNA molecule related to a specific hereditary trait is called a gene.
• Plasmids can also be used as vectors to carry new genetic material into a host organism. This is accomplished by first cutting the plasmid with an enzyme, known as a restriction endonuclease, that opens the circle of DNA. A fragment of foreign DNA, containing the desired gene, is inserted into the place where the DNA circle was cut. The circle is reformed by treatment with DNA ligase.
• an enzyme known as a restriction endonuclease
• the recombined plasmid a new circular DNA molecule, contains the genes of the original plasmid plus the new gene from the piece of DNA which was inserted.
• This plasmid can be introduced into a host microorganism.
• the plasmid containing the new gene is then reproduced in the host microorganism and becomes part of its genetic material.
• microorganism For a host microorganism to be suitable for use in genetic engineering, it must be capable of incorporating the new DNA. Furthermore, it must yield a viable microorganism which expresses the traits coded in the newly inserted gene. For the microorganism to produce useful quantities of protein, the microorganism must also be one that can be grown on a commercial scale.
• Biological containment relates to the use of host cells and vectors which have limited ability to survive if they escape from the laboratories into the natural environment.
• Cells of microorganisms which do not form spores i.e., ones that are asporogenous have such a limited ability to survive.
• the present invention describes a new asporogenous mutant of B . subtilis Bl-109 useful as host in a host-vector system.
• This host has the additional advantage of being an amylase-negative mutant.
• the mutant is particularly suitable for use as a host for recombinant plasmids which contain genes coding for the production of specific amylases such as thermostable alpha -amylases. These amylases are of commercial importance for use in processes to make starch hydrolyzates, glucose, and high fructose syrups.
• the mutant of the present invention readily incorporates plasmids containing a thermostable alpha -amylase gene.
• the resulting microorganism is a superior producer of thermostable alpha -amylase enzyme to host-vector systems prepared from previously described amylase-negative strains of B . subtilis such as ATCC 39,096, disclosed in published European Patent Application No. 092 235, issued on October 26, 1983.
• a culture of asporogenous B . subtilis Bl-109 suitable for use as a host component in a host-vector system characterized in that it contains no amylase-coding gene, has a frequency of reversion to spore formers of less than about 10 ⁇ 7 when grown under conditions of aeration and having the following genetic markers: spoIIA 12, amyE , and SacA 321.
• a strain of B . subtilis having these characteristics has been deposited with the American Type Culture Collection as ATCC 39,701.
• Also provided in accordance with this invention is a process for preparing a strain of B . subtilis having the genetic markers: metB 5, amyE, and sacA 321, which comprises transforming competent cells of B . subtilis Strain lA289 with DNA from B . subtilis Strain lA221 and isolating cells that do not produce alpha-amylase enzyme.
• a process for preparing a strain of B . subtilis having the genetic markers: spoIIA 12, amyE , and sacA 321, which comprises transferring competent cells of B . subtilis Strain Bl-20 with DNA from B . subtilis strain lS30 and isolating cells that grow in the absence of added methionine, do not produce alpha -amylase enzyme, and that do not form spores when heat shocked at 90°C for 10 minutes.
• the B . subtilis strains disclosed and claimed were prepared by incorporating the genetic material from other strains of B . subtilis .
• An amylase-negative strain of B . subtilis IA289 (ATCC 39,711) containing the markers: aroI 906, metB 5, sacA 321, and amyE was transformed into a strain no longer requiring aromatic amino acids by incorporation of a portion of the DNA from B . subtilis Strain lA221 (ATCC 39,086).
• Strain lA221 was reported by Dubnau and Smith, Proc. Natl. Acad. Sci. , U.S.A., 65 , 96-103 (1970). It is available from the American Type Culture Collection, Rockville, Maryland, as ATCC 39,086.
• the resultant transformant designated as Bl-20, contains the markers: metB 5, amyE , and sacA 321. It is available from the American Type Culture Collection as ATCC 39,706.
• This amylase-negative strain is useful as an intermediate for the preparation of amylase-negative host components of host-vector systems. It can also serve as a host itself under conditions where a spore-forming host is acceptable.
• a portion of the DNA from an asporogenic strain of B . subtilis was transferred into Strain Bl-20 to produce new Strain Bl-109.
• the asporogenic strain employed as the DNA donor was lS30 obtained from the Bacillus Genetic Stock Center, Dept. of Microbiology, Columbus, Ohio. This strain which contains the marker spoIIA 12, was described by Ionesco and Schaeffer, Ann. Inst. Pasteur, Paris, 114 , 1-9 (1968). It is available from the American Type Culture Collection, Rockville, Maryland, as ATCC 39,712.
• the asporogenous, amylase-negative strain of the present invention shows a frequency of reversion to spore formers of less than about 10 ⁇ 7. It is able to grow under industrial conditions and does not require expensive growth additives. It has a low survival rate under natural or escape conditions and a very low tendency to transmit plasmids to other organisms by natural genetic transfer.
• the organism shows a high degree of competence for plasmid uptake when subjected to classical transformation techniques. Excellent transformations were achieved using competent cell transformation procedures. It functions well as a host for various plasmid vectors making it useful as a host component of a B . subtilis host-vector system. Being amylase-negative, it is particularly suitable for use as a host for recombinant plasmids which contain genes coding for the production of amylases.
• the cells were separated by centrifugation and suspended in 5 volumes of a growth medium containing 0.5% glucose, 0.6% KH2PO4, 1.4% K2HPO4, 0.1% sodium citrate, 0.2% (NH4)2SO4 and 0.07% MgSO4 supplemented with leucine, isoleucine and methionine at a level of 50 ⁇ g/ml and with histidine, tryptophan, arginine, valine, lysine, threonine, glycine and aspartic acid at a level of 25 ⁇ g/ml.
• the increase in optical density of the culture at 620 nm was monitored using a spectrophotometer. When cultures reached the transition between log and stationary growth (change in optical density of less than 5% in 15 minutes), they were used for transformation with DNA.
• Donor DNA was obtained from B . subtilis Strain lA221 (ATCC 39,086) by the following procedure.
• Strain lA221 was grown in 100 ml Penassay Broth R (Difco). The mixture was grown at 37°C until the optical density of the mixture measured at 660 nanometers was 0.6. The cells were collected by centrifugation and resuspended in 1 ml of a solution containing 0.005 M ethylenediamine tetraacetic acid R (EDTA) and 0.05 M NaCl containing 2 mg of lysozyme. When the cells began to lyse, they were frozen by immersing their container in a dry ice-acetone bath.
• EDTA ethylenediamine tetraacetic acid
• Competent cells of B . subtilis Strain lA289 (1.0 ml) prepared as described above, were mixed with DNA at a concentration of 10 ⁇ g/ml of final mixture and the culture was shaken gently (100 rpm) for 30 minutes at 37°C. The culture was then diluted with 2 ml of Penassay Broth R (Difco) and allowed to grow for an additional 90 minutes at 37°C. The cells were collected by centrifugation, washed once with distilled water, resuspended in the original volume of medium and spread on plates containing agar with Spizizen's minimal medium supplemented with methionine (5 ⁇ g/ml).
• Spizizen's minimal medium is a solution of (a) ammonium sulfate 0.2%; (b) potassium phosphate (dibasic) - 1.4%; (c) potassium phosphate (monobasic) - 0.6%; (d) sodium citrate - 0.1%; and (e) magnesium sulfate - 0.02%; pH adjusted to 7.4.
• ammonium sulfate 0.2% potassium phosphate (dibasic) - 1.4%
• potassium phosphate (monobasic) - 0.6% potassium phosphate (monobasic) - 0.6%
• magnesium sulfate - 0.02% pH adjusted to 7.4.
• Strain B1-20 metB 5, amyE , sacA 321 was converted into the asporogenous strain, B1-109, by transformation with DNA from the donor strain, 1S30 (ATCC 39,712) . Transformation was carried out using competent B1-20 cells.
• Donor DNA was obtained from B . subtilis Strain 1S30 (ATCC 39,712) by the following procedure. Cells were grown overnight at 37°C in 100 ml of a Tryptic Soy with glucose (TSG) medium R (Difco). Then 40 mg of lysozyme was added, and the mixture was incubated for 30 minutes at 60°C. The spheroplasts were separated by centrifugation for 10 minutes at 4500 rpm and suspended in 40 ml of a solution containing 0.15 M NaCl and 0.1 M EDTA at pH 10.2. After 3 ml of 25% sodium dodecyl sulfate solution was added, the mixture was incubated at 70°C for 1 hour.
• TSG Tryptic Soy with glucose
• the protein was precipitated twice by additions of a solution containing 0.03 M NaCl and 0.003 M sodium citrate at pH 7 saturated with phenol. To the supernatant was added 5 ml of 3 M sodium acetate solution. A layer of isopropanol was added to the solution. DNA which formed at the interface was wound onto a glass rod. The DNA on the rod was washed twice with cold isopropanol and dissolved in a solution containing 0.03 M NaCl and 0.003 M sodium citrate at pH 7. The resulting solution was dialyzed against three changes of a large volume of a solution containing 0.015 M NaCl and 0.0015 M sodium citrate at pH 7 during a 24-hour period before it was used for transformation.
• B . subtilis Strain Bl-20 were grown at 37°C overnight on plates containing Tryptose Blood Agar Base R (Difco). Cells from the plates were then suspended in a small amount of growth medium and used to inoculate about 60 ml of additional growth medium in a 500-ml Erlenmeyer flask. Mixture was grown at 33-37°C with shaking.
• the growth medium contained the following ingredients: 0.5% glucose, 0.6% KH2PO4, 1.4% K2HPO4, 0.1% sodium citrate, 0.2% (NH4)2SO4, 0.01% MgSO4, 0.02% Casamino acids (Difto), 0.1% yeast extract (Difco) and 0.02% L-tryptophan.
• Transformation was accomplished by mixing 20 ⁇ l of DNA isolated from Strain lS30 as described above with 0.5 ml of competent cells of Strain Bl-20 described above. The mixture was incubated at 30-37°C for 90 minutes with shaking at 250 rpm. The cultures were plated on agar plates after dilution with sterile water. Cells showing methionine-positive, amylase-negative characteristics were selected by growing cultures on agar plates containing Spizizen's minimal medium supplemented with tryptophan (5 ⁇ g/ml) and 1% Lintner starch. Six hundred colonies were then screened for asporogenic characteristics by heat shocking them for 10 minutes at 90°C. One colony, Bl-109, was asporogenic. It has the genetic markers: spoIIA 12, amyE , and sacA 321. A biologically pure culture of this strain is on deposit at the American Type Culture Collection as ATCC 39,701.
• Strain Bl-109 requires mineral salts containing ammonium, potassium, phosphate and sodium ions for growth. It will utilize various carbon sources including glucose. The strain does not revert to spore formers as shown by the following test. Cells were grown on agar plates containing DSM R (Difco) medium at 37°C. After 24 hours, the cells were suspended in a tryptic soy medium containing 0.1% glucose and heated at 90°C for 10 minutes. The cells were then titered on plates containing Tryptose Blood Agar Base R (Difco). Total viable cells were determined as colony-forming units on these plates. Comparisons were made with a number of colony-forming units per milliliter developed when unheated samples were titered.
• DSM R Difco
• the unheated samples contained 1.27 x 108 colony-forming units per ml, whereas, the heated cells contained only 5 colony-forming units per ml. This indicates that the strain has a frequency of reversion to spore formers of less than about 10 ⁇ 7 when grown under conditions of aeration.
• the plasmid vectors are retained in these hosts even when the hosts with the vectors are grown at temperatures as high as 45°C.

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• 1985
  • 1985-04-30 IE IE109385A patent/IE58099B1/en not_active IP Right Cessation
  • 1985-05-10 CA CA000481232A patent/CA1288710C/en not_active Expired - Fee Related
  • 1985-05-22 FI FI852038A patent/FI81380C/fi not_active IP Right Cessation
  • 1985-05-29 AU AU43086/85A patent/AU4308685A/en not_active Abandoned
  • 1985-05-30 BR BR8502586A patent/BR8502586A/pt unknown
  • 1985-06-04 DK DK251385A patent/DK251385A/da not_active Application Discontinuation
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  • 1985-06-05 EP EP85106994A patent/EP0164117B1/en not_active Expired - Lifetime
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  • 1985-06-06 JP JP60121627A patent/JPS611381A/ja active Granted
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